The Growth Hormone Releasing Hormone Signaling Pathway Governs Cardiomyocyte differentiation in human iPS cells.

Differentiation and expansion of cardiomyocytes from human induced pluripotent stem cells is a poorly understood process, and the signals promoting differentiation of cardiomyocytes toward specific cell fates are not well defined. Here we show that the growth hormone-releasing hormone receptor (GHRH-R) signaling pathway controls this differentiation process and cell fate. Stimulation of hPSCs with GHRH promotes β-catenin stabilization through the GH/IGF1 axis. The presence of cell-surface GHRH-R differentiates between atrio-ventricular cardiomyocytes and non-cardiomyogenic cardiac cells, including coronary vascular, neuronal and pacemaker cells, such that selecting for GHRH-R allows for the elimination of pacemaker cells and derivation of a pure population of atrio-ventricular cardiomyocytes. Unlike SIRPA, cardiac GHRH-R identifies murine and human NKX2-5+ myocardial derivatives of both mesodermal and neuroectodermal lineages. Using RNA-seq to comprehensively characterize the GHRH-R signaling pathway, we identified that NKX2-5, a transcription factor involved in progenitor specification and differentiation into atrio-ventricular cardiomyocytes, is upregulated in response to GHRH. GHRH-R signaling also upregulated HIF-1α via the PI3k/Akt/mTOR pathway to enhance NKX2-5 transcription and stimulate a Warburg-like effect in developing cardiomyocytes. Together these findings provide crucial insights into human stem cell differentiation and offer a translationally useful tool to optimize the development of pure cardiomyocyte preparations derived from induced pluripotent stem cells. ### Competing Interest Statement The authors have declared no competing interest.